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目的观察核转录因子-κB(NF-κB)圈套寡核苷酸对NF-κB活性的影响,研究Survivin在其诱导肝癌细胞HepG2凋亡中的作用。方法将异硫氰酸荧光素(FITC)标记的NF-κB圈套寡核苷酸转染至HepG2细胞中,荧光倒置显微镜和共聚焦显微镜进行核内定位观察,凝胶迁移率实验(electrophoretic mobility shift assay,EMSA)检测转染前后NF-κB活性变化;流式细胞仪和TUNEL检测细胞凋亡率;Western blot检测凋亡抑制蛋白Survivin的改变。结果NF-κB圈套寡核苷酸转染后定位于细胞核内,EMSA显示转染后NF-κB活性明显下降。与未处理组、转染错义组相比,转染NF-κB圈套寡核苷酸组的HepG2细胞凋亡率增加,Survivin蛋白表达下调[(39.8±1.9)、(42.7±2.5)vs(20.1±3.1)]。结论NF-κB圈套寡核苷酸具有促进肝癌细胞HepG2凋亡的作用,其机制可能与下调Survivin的表达有关。
Objective To investigate the effect of nuclear factor-kappaB (NF-κB) truncated oligonucleotide on the activity of NF-κB and the role of survivin in inducing HepG2 cell apoptosis. Methods FITC-labeled NF-κB decoy oligonucleotides were transfected into HepG2 cells. Fluorescent inverted microscopy and confocal microscopy were used for nuclear localization observation. The electrophoretic mobility shift assay, EMSA) before and after transfection NF-κB activity changes; flow cytometry and TUNEL detection of apoptosis rate; Western blot detection of apoptosis inhibitor protein Survivin changes. Results NF-κB decoy oligonucleotides were located in the nucleus after transfection. EMSA showed that NF-κB activity was significantly decreased after transfection. The apoptosis rate of HepG2 cells transfected with NF-κB decoy oligonucleotide group was significantly higher than that of the untreated group (P <0.05), and the expression of Survivin protein was down-regulated [(39.8 ± 1.9) vs (42.7 ± 2.5) vs 20.1 ± 3.1)]. Conclusion NF-κB decoy oligodeoxynucleotides can promote the apoptosis of HepG2 hepatocarcinoma cells, which may be related to the down-regulation of Survivin expression.